The Disulfide Folding Pathway of Human Epidermal Growth Factor

Abstract Human epidermal growth factor (EGF) contains three disulfides and 53 amino acids. Reduced/denatured EGF refolds spontaneously in vitro to acquire its native structure. The mechanism of this folding process has been elucidated by structural analysis of both acid and iodoacetate trapped intermediates. The results reveal that the folding is accompanied by a sequential flow of unfolded EGF (0-disulfide) through three groups of folding intermediates, namely 1-disulfide, 2-disulfide, and 3-disulfide (scrambled) EGF isomers, to reach the native structure. Equilibrium occurs among isomers of each class of disulfide species, and the composition of intermediates appears to be highly heterogeneous. Together, at least 27 fractions of folding intermediates have been identified, but there exist only limited numbers of well populated species which constitute more than 80% of the total intermediates found during EGF folding. Six species of such well populated intermediates have been isolated, which included two 1-S-S, two 2-S-S, and two 3-S-S scrambled species. Their disulfide structures have been identified here. Both 1-S-S isomers are found to contain non-native disulfides. One of the 2-S-S species consists of two non-native disulfides and the other admits two native disulfides. Among the six disulfides of the two scrambled species, only one is native. Together, native disulfides constitute 25% of the total disulfides found in these six well populated intermediates. These results contrast sharply to those observed with bovine pancreatic trypsin inhibitor, which has shown that well populated folding intermediates consist of exclusively native disulfides (Weissman, J. S., and Kim, P. S. (1991) Science 253, 1386-1393). We propose that well populated folding intermediates, regardless of whether they contain native or non-native disulfides, do not necessarily represent the productive species and specify the folding pathway. Furthermore, conditions influencing the efficiency of EGF folding have been investigated. It is demonstrated here that under optimized compositions of redox agents, including the use of cysteine/cystine and protein disulfide isomerase, the in vitro folding of EGF could be achieved quantitatively within 1 min.